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I'm using cuffdiff2 to examine the behavior of two paralogs in different maize haplotypes, and I've run into some output that isn't consistent withe previous data on these genes. qRT-PCR on these genes shows expression and Tophat alignments of single-end Illumina reads show expression as well. However, when I run cuffdiff2 on these files (2 biological replicates per haplotype), gene1 is assigned non-zero FPKM values and gene2 is assigned zero FPKM in all haplotypes. The quantification status for both genes is OK in all instances. I've had issues before with cuffdiff not recognizing annotated genes, creating novel transcripts where annotated models exist, etc., so I looked for a "novel" transcript at the same locus as gene2 but found nothing. I'm rather stymied at this point, and I'm curious if it sounds like I'm doing something wrong, others are having a similar problem, how to fix it, suggestions.
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I’m not a sequencing expert. I’m a purification scientist who uses NGS to evaluate workflows my group develops. With this perspective, we think about the sample first and the NGS workflow second. The sequencer is an exceptionally honest reporter, but it can only report on what you give it, so whether you get clean, interpretable data from an NGS workflow is largely determined before you begin.
Here are nine questions we think about, in roughly the order they matter, before...06-18-2026, 07:11 AM -
Data variability is still an issue in sequencing technologies despite the advances in reproducibility and accuracy of these platforms. But the problem does not originate in the sequencing itself, but in the previous steps, before the sample reaches the sequencer.
The first step is collection, followed by preservation and sample preparation for analysis. Most scientists overlook those steps, but not being careful might just be skewing the experiment’s results.
...06-02-2026, 10:05 AM